Glycogen levels modulate myocardial glucose uptake.

Abstract

Glucose uptake is mediated by a family of glucose transporters [ 11, predominately GLUTl and GLUT4 in the heart. GLUTl is present solely in the plasma membrane and is primarily responsible for basal glucose uptake [2], whereas insulin stimulated glucose transport is mediated by GLUT4 [3]. In the heart, insulin increases glucose transport by increasing the Vmax of the glucose transporter [4], via a mechanism that involves increased translocation of GLUT4 from an intracellular vesicular pool to the plasma membrane [5]. The rate of glucose uptake can also be modulated by other changes in the heart unrelated to the insulin action. Increased glycolysis causes increased glucose uptake, for example in ischaemia [6], anoxia [7] or during increased cardiac work load [8]. The mechanism is believed to involve GLUT4 translocation to the plasma membrane by a mechanism distinct from that of insulin action [8-lo]. The. influence of glycogen metabolism on glucose uptake is less well understood, but glycogen is another site for glucose disposal in the myocyte, in addition to glycolysis. As increasing glycolytic flux can result in increased glucose transport, we hypothesize that the demand for glucose to be incorporated into glycogen may also regulate glucose uptake. To clarify the relationship between myocardial glycogen levels and glucose uptake, we measured basal and insulin stimulated uptake in the isolated rat heart having acutely altered glycogen levels, using both 2DG and [2-’H]glucose uptake. Hearts from male Wistar rats (body weight 350 400 g) were perfused using the Lmgendorff method at a constant pressure of 100 mmHg at 37’C. The perfusate was modified Krebs-Henseleit buffer that contained 112 mM NaCI, 4.7 mM KCI, 1.2 mM MgSOe7H20,1.75 mM CaC1,.2H20, 0.5 mM N%EDTA. 25 mM NaHCO,, 1.2 mM KH,PO,, equilibrated with 95% 0, / 5% CO, to give a pH of 7.4 at 37°C. Glycogen levels were modified prior to uptake measwments by perfusion with different substrates for 30 minutes; 5 mM pyruvate, 1 mM acetate and 1 mM glucose for controls; 4 mM 8-hydroxybutyrate, 1 mM acetoacetate and 11 mM glucose for high glycogen; 11 mM glucose and 20 nM isoproterenol for low glycogen. Basal glucose uptake was measured following glycogen modification by perfusion for 36 min with a buffer containing 5 mM pyruvate, 1 mM acetate and 2 mM GM. Insulin was then added at 2.86 mU/ml, levels sufficient to give maximum stimulation of glucose transport, for a further 20 min perfusion. 2DG uptake and phosphorylation was assessed using ”P NMR spectroscopy. The heart and cannula system were placed in a 20 mm NMR tube, which was then inserted into the bore of a 9.4 T superconducting magnet. ”P NMR spectra were acquired using a Bruker AM-400 NMR spectrometer, 60” pulse and 104 s c y s with a recycle h e . of 2.15 s, giving 4 min time resolution. P NMR spectral peaks were fitted to Lorentzian line shapes using NMRl (NMRi, Syracuse, USA) and the peak areas quantified. An external standard containing 10 pmoles of phenylphosphonic acid was used to verify quantification of phosphate metabolites. The pH was estimated from the chemical shift of the Pi peak relative to the PCr peak using a titration c w e at 1 mM [Mg”], at 37°C. In a separate set of experiment, [2-3H]glucose was used as the marker for glucose uptake. Hearts were perfused initially at 100 mmHg pressure using non-recirculating perfusion buffer (as described above). After alteration of glycogen levels with different substrates the hearts were perfused with 300 ml of recirculating perfusion buffer [ l l ] at 100 mmHg constant pressure. This recirculating buffer contained 2 mM glucose plus 20 pCi [23H]glucose as the GM [12]. Samples (0.5 ml) of the perfusion buffer were taken eve? 4 min, to coincide with the NMR measurements, and the H,O separated from the [2-’H]glucose using an ion exchange column and counted using liquid scintillation. Glycogen levels were modified without any significant changes in ATP, PCr. pH or G6P (Table 1). Myocardial function was also unaltered in hearts with different glycogen levels. Basal 2DG uptake was increased 2-fold in low glycogenand decreased 39% in high glycogencontaining hearts. Insulin sensitive 2DG transport was similarly increased 1.6-fold in low glycogenand decreased 20% in high glycogen-containing hearts (Table 2). [2-3H]glucose